Project Summary Phospholipid nanodiscs have attracted great interest over the last two decades as a means to provide a native- like bilayer environment for study of embedded membrane proteins. A popular version is assembled with two copies of membrane-scaffold proteins (MSPs), derived from Apolipoprotein A1, that form a double-layer belt around the hydrophobic perimeter of a lipid patch about 10 nm in diameter. We recently reported recombinant versions of MSP that, after cyclization with sortase, enable assembly of nanodiscs up to 80 nm in diameter. However, the largest ones are prone to fusion and aggregation. Here we propose to construct DNA-origami corrals that direct the reconstitution of multiple 10 nm MSP nanodiscs into larger ones from 60 nm to 1000 nm in diameter. DNA corrals additionally act as bumper cases to prevent unwanted aggregation and can enable control over stoichiometry, geometry, and orientation of inserted guests through tethering to the corral. We will investigate clusters of medium-sized (60 nm diameter) nanodiscs, each with a varying tilt angle, and large-sized (>200 nm diameter) nanodiscs for capture for a high density of guest membrane proteins, in either noncrystalline or crystalline arrangement, for cryoEM analysis. A major benefit will be in mediating faster data collection through presentation of high-density of guests. Presentation in crystalline format could reduce conformational variability, which can lead to improved particle classification. The primary objectives of this proposal are as follows: In Aim 1, we will generate DNA-corralled nanodiscs (DCNDs) from 60 nm up to 1 ?m in diameter for hosting lipid- embedded membrane proteins at high density. We will investigate DNA-origami ?barrels? with outer modifications that enable lateral clustering, inner modifications that enable free rotation of guest MSP nanodiscs, and inner modifications that enable assembly of double-decker MSP nanodiscs. We will investigate DNA-origami ?arenas?, composed as self-limiting homopolymers of rigid V-shaped wedges, that enclose MSP nanodiscs with a diameter from 200 nm to 1000 nm. In Aim 2, we will optimize protocols for reliable embedding of large membrane proteins and their complexes in DNA-corralled nanodiscs. In particular, we will engineer DNA- corralled SMA nanodiscs and use them to extract VDAC/ANT1/HK complex from native mitochondrial membranes. We also will engineer asymmetric, DNA-corralled nanodiscs with respect to lipid distribution using different subtypes of flippases positioned on one face. In Aim 3, we will use cryoEM for structural analysis of hosted membrane proteins and their complexes in DCNDs. (i) We will embed complexes of VDAC-1 with hexokinases, creatine kinases and ANT1 in DCNDs and study their structures with electron microscopy techniques. (ii) We will place multiple copies of CCR5 and CD4 in DCNDs containing asymmetric bilayers matching the immune cell membrane to study the interaction with the HIV1 gp160 or smaller constructs. (iii) We will use the same DCND decorated with co-receptors to image the interaction with non-infectious HIV-1 virus- like particles (VLPs). Negative stain and cryoEM will be used to image the systems of interest.